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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Correction, I meant 0.05% tricaine.
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Best,
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<br>
T<o:p></o:p></span></p>
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<p class="MsoNormal"><b><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif"">From:</span></b><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif""> Jan Huisken [mailto:huisken@mpi-cbg.de]
<br>
<b>Sent:</b> Friday, April 29, 2016 3:01 AM<br>
<b>To:</b> Feinstein, Timothy N<br>
<b>Cc:</b> openspim@openspim.org<br>
<b>Subject:</b> Re: [OpenSPIM] Is anyone using digital scanning to reduce banding?<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Dear Tim,<o:p></o:p></p>
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<p class="MsoNormal">I think you are mixing up two issue.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><b>1. Getting rid of stripes.</b><o:p></o:p></p>
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<p class="MsoNormal">Yes, you can do a little bit in post-processing but the stripes can be quite complex and are not necessarily straight. Oftentimes you see a lot of lensing and I do not think you can easily remove it with image processing.<o:p></o:p></p>
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<p class="MsoNormal">a) Double-sided illumination helps. Depending on the sample you may want to do simultaneous or sequential double-sided illumination. There are various ways of merging the two images.<o:p></o:p></p>
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<p class="MsoNormal">b) A resonant mSPIM mirror is cheap and easy to integrate. You will get rid of most stripes, but again this depends on the sample. The details for a) and b) are in our Opt. Lett. paper (Huisken & Stainier, 2007). Basically, you are pivoting
the light sheet around the center of your field of view, e.g. with 1kHz. The stripes are “washed out” during the exposure time of your camera. We use this on most of our systems and it helps a lot to reduce stripes.<o:p></o:p></p>
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<p class="MsoNormal">c) Multi-view fusion can also help to some extent.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><b>2. DSLM vs. SPIM</b><o:p></o:p></p>
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<p class="MsoNormal">Yes, DSLM should also help reducing stripes but I have no experience with that. Basically, your light sheet is not coherent anymore and the sheet does not “interfere with itself”. However, the setup is more expensive and requires you to
power and control another element: the galvo scanner to sweep the beam up and down. You need a proper scan mirror and not just the cheap mSPIM mirror. The additional benefit is that the resulting light sheet is more uniform than the cropped Gaussian light
sheet. However, you need to illuminate each line with higher intensity which may result in saturation and worse dynamic range. Obviously you need to wait for the scan and need to synchronize your readout. Depending whether you have a global shutter or rolling
shutter camera you need to take some precaution.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Best<o:p></o:p></p>
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<p class="MsoNormal">Jan<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="color:black">—<o:p></o:p></span></p>
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<p class="MsoNormal"><b><span style="color:black">Jan Huisken</span></b><span style="color:black"><o:p></o:p></span></p>
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<p class="MsoNormal"><i><span style="color:black">Head of Max Planck Research Group</span></i><span style="color:black"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="color:black"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="color:black">Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)<o:p></o:p></span></p>
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<p class="MsoNormal">On Apr 27, 2016, at 4:13 PM, Feinstein, Timothy N <<a href="mailto:tnf8@PITT.EDU">tnf8@PITT.EDU</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal">Hello all, <br>
<br>
I found the Selzer group's solution (Keller et al. 2013) quite interesting<br>
and would love to know if anyone had a positive experience doing that.<br>
The banding from my basic openSPIM is definitely a problem for<br>
segmentation and analysis.<br>
<br>
As far as I can tell fixing the banding will mostly involve buying a scan<br>
mirror. I don't have a quote yet for the Cambridge VM500 scanning mirror<br>
that the Selzer group used but its ThorLabs equivalent (#GVS101?) costs<br>
about $1k. That seems reasonable and not that hard for regular folks to<br>
implement. <br>
<br>
In related news I just noticed that the TeraStitcher plugin for Vaa3D now<br>
has an option to fight banding in light sheet data. I have not tested it<br>
yet but potentially that feature could be useful. I will just post my<br>
usual warning for folks trying TeraStitcher for the first time: it<br>
stitches really well but it could be a hair more user-friendly.<br>
<br>
Best, <br>
<br>
<br>
Tim<br>
<br>
Timothy Feinstein, Ph.D.<br>
Research Scientist<br>
University of Pittsburgh Department of Developmental Biology<br>
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<br>
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